New competence stimulating peptide

ABSTRACT

The present invention concerns a new competence stimulating peptide identified in Firmicutes, in particular  Streptococcus , and more preferably  S. thermophilus  and methods of producing transformation competent Firmicutes, in particular  Streptococcus , and more preferably  S. thermophilus  bacteria.

DOMAIN OF THE INVENTION

The present invention relates to Firmicutes, in particularStreptococcus, and more preferably S. thermophilus competence, and morespecifically to a new competence stimulating peptide.

BACKGROUND OF THE INVENTION

Many species of bacteria control gene expression on a community-widescale by producing, secreting, detecting and responding to extracellularsignaling molecules (sometimes called ‘autoinducers’ or ‘pheromones’)that accumulate in the environment. This phenomenon is termed ‘quorumsensing’ (QS) as gene expression is triggered by the ‘sensing’ of thepheromone when its concentration has reached a ‘quorum’. InGram-positive bacteria, the signaling molecules are mainly shortpeptides acting either from the outside part of bacteria or from theinside, after internalization via oligopeptide transport systems calledOpp or Ami.

Several bacterial functions such as the virulence in Staphylococcusaureus and Enterococcus faecalis, the competence in Bacillus subtilis orthe production of bacteriocin in Lactococcus lactis are controlled bypeptides acting at the surface of bacteria. However, the paradigm formechanisms involving peptides detected from the outside is theregulation of the competence state for natural transformation bychromosomal donor DNA in Streptococcus pneumoniae. In this bacterium,the signaling peptide, called CSP (Competence Stimulating Peptide) andencoded by the gene corn C, is secreted and matured by an ABCtransporter, ComAB. The detection of the extracellular CSP at thesurface of the bacterium is achieved by a two component system (TCS).Firstly, the membrane embedded histidine kinase, ComD,autophosphorylates in response to CSP and further phosphorylates itscognate response regulator, ComE, which activates transcription of a fewgenes, the early CSP-induced genes, including comCDE, comAB and comX.ComX is an alternative competence specific sigma factor required forexpression of late CSP-induced genes, which comprise genes encoding theDNA uptake machinery.

Concerning signaling peptides that are active after internalization byan oligopeptide transporter, three groups have been described in detail:(i) Phr peptides in B. subtilis involved in the control of sporulation,competence, and production of degradative enzymes and antibiotics, (ii)PapR peptides involved in the control of virulence of bacteria belongingto the Bacillus cereus group and (iii) peptides involved in the controlof plasmid transfer in Enterococcus faecalis. All these extracellularshort peptides interact with either Rap phosphatases (in B. subtilis) ortranscriptional regulators (PieR in B. cereus or PrgX in E. faecalis) toelicit a physiological response.

Oligopeptide transport systems involved in these signaling pathwaysbelong to the superfamily of ATP-binding cassette (ABC) transporters.They are composed of five subunits: an extracellularoligopeptide-binding protein, OppA that specifically captures thesubstrates, two transmembrane proteins, OppB and OppC that form the poreand two membrane-bound cytoplasmic ATP-binding proteins, OppD and OppFthat provide the energy for peptide translocation. Several copies of theopp operon and/or of the genes encoding the oligopeptide-bindingproteins can be present in a single genome. The genome of Streptococcusthermophilus encodes one oligopeptide transport system and depending onthe strain, two (strain LMD-9 and CNRZ1066) or three (strain LMG18311)oligopeptide-binding proteins. In Gram-positive bacteria, two mainfunctions have been attributed to the Opp transporters: nutrition andsensing. The nutritional role has been well studied in lactic acidbacteria such as Lactococcus lactis or S. thermophilus. During growth inmilk, the Opp transporters supply these auxotrophic bacteria withpeptides that serve as amino acid sources. The sensing function is morecomplex and is poorly documented, particularly in nonpathogenicbacteria.

Among the nonpathogenic bacteria, S. thermophilus is of major importancefor the food industry since it is massively used for the manufacture ofyoghurt and Swiss or Italian-type cheeses with an annual market value ofapproximately $40 billion making S. thermophilus a species of majoreconomic importance. The industry is continuously working to improve theproperties of S. thermophilus starter strains. Even though thefermentation properties of this bacterium have been gradually improvedby classical methods, there is great potential for further improvementthrough genetic engineering.

However, until now, only genetic tools based on genetically modifiedbacteria exist. For example, Havarstein has disclosed an induciblesystem (stb system) that permits the surexpression of proteins in S.thermophilus (Blomgvist T. et al “Pheromone-induced expression ofrecombinant proteins in Streptococcus thermophilus” Arch Microbiol. 2006December; 186(6):465-73. Epub 2006 Aug. 24). In particular, this articlediscloses that a possible peptide-pheromone (STP) regulates bacteriocinproduction in S. thermophilus LMG 18311, and shows that the StbABCHR(system that regulates bacteriocin production) quorum-sensing system canbe exploited for inducible expression of recombinant proteins in thisbacterial species.

Thus, there is a need for an efficient method that allows obtainingimproved Firmicutes, in particular Streptococcus, and more preferably S.thermophilus by natural processes of gene transfer, and not artificialgene transfer. This is particularly important for the food industries,for example the dairy industries which do not want to use GMO in theirproducts.

Regarding this need of genetic tools, competence is poorly understood inS. thermophilus. In fact, regarding QS systems, only one of them hasbeen yet described, which QS system (called stb or blp) controls theproduction of a bacteriocin.

The sequencing of the genome of three strains of S. thermophilus,CNRZ1066, LMG18311 and LMD-9, has revealed the presence of comX and of14 proteins with strong similarities with the 14 proteins known to berequired for competence in S. pneumoniae and encoded by late CSP-inducedgenes (Bolotin et al., Complete sequence and comparative genome analysisof the dairy bacterium Streptococcus thermophilus, Nat. Biotechnol.22:1554-1558, 2004; Makarova et al. Comparative genomics of the lacticacid bacteria. Proc Nall Acad Sci USA., 103(42):15611-6, 2006). ExceptcomX, no ortholog of the early CSP-induced genes of S. pneumoniae havebeen detected in the genome of S. thermophilus. It has been shown thatoverexpression of comX induces the competent state in S. thermophilusLMG18311.

Still, how transformation is turned on in this strain and whatregulatory pathway and more especially which competence stimulatingpeptide (CSP) controls the expression of comX have not previously beenexplained.

SUMMARY OF THE INVENTION

The present invention relates to an isolated polypeptide comprising theamino acids sequence SEQ ID No 1 (LKTLKIFVLFSLLIAILPYFAGCL), orderivatives or fragments thereof capable of stimulating competence inFirmicutes, in particular Streptococcus, and more preferably S.thermophilus.

The present invention also relates to an isolated polypeptide comprisingthe amino acid sequence SEQ ID No 7 (IAILPYFAGCL) or derivatives orfragments thereof capable of stimulating competence in Firmicutes, inparticular Streptococcus, and more preferably S. thermophilus.

The present invention also relates to an isolated nucleic acid encodingfor said isolated polypeptides.

The present invention still relates to a vector comprising said nucleicacid operably linked to a gene expression sequence.

The present invention still relates to a host cell geneticallyengineered with said vector.

The present invention also relates to a culture medium comprising aneffective amount of said isolated polypeptide and nutrients that allowthe growing of Firmicutes, in particular Streptococcus, and morepreferably S. thermophilus.

The present invention further relates to a use of said polypeptide, saidnucleic acid, said vector, said host cell, or said culture medium forstimulating competence in Firmicutes, in particular Streptococcus, andmore preferably S. thermophilus.

The present invention still relates to a method of producingtransformation competent Firmicutes, in particular Streptococcus, andmore preferably S. thermophilus bacteria comprising the step (i) ofcontacting said Firmicutes, in particular Streptococcus, and morepreferably S. thermophilus bacteria with an effective amount of anisolated polypeptide as defined in any one of claims 1 to 5 forobtaining said transformation competent Firmicutes, in particularStreptococcus, and more preferably S. thermophilus bacteria.

DESCRIPTION OF THE FIGURES

FIG. 1: Development of competence during growth of strain LMD-9 in CDMusing pG⁺host9 plasmid as transformant DNA.

FIG. 2: Development of competence during growth of strain LMD-9 in CDMusing chromosomal DNA of strain TIL1192 as transformant DNA.

FIG. 3: Relative expression levels of comX, recA, dprA and comGA betweenS. thermophilus LMD-9 and strain TIL883 (LMD9 ΔamiCDE) or strain TIL1196(LMD-9 comX::erm).

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, the inventors have established that the Ami oligopeptidetransport system is implicated in the control of competence inStreptococcus, in particular S. thermophilus. This transport systemfunctions with several oligopeptide binding proteins. In strain LMD9where two oligopeptide binding proteins are present, AmiA3 and AmiA1,AmiA3 plays the major role in the control of competence.

More specifically, the inventors have identified one new polypeptide andfragments thereof implicated in the control of competence.

In a first aspect, the present invention relates to an isolatedpolypeptide comprising the amino acids sequence SEQ ID No 1(LKTLKIFVLFSLLIAILPYFAGCL), or derivatives or fragments thereof capableof stimulating competence in Firmicutes, in particular Streptococcus,and more preferably S. thermophilus.

The isolated polypeptide of the invention has the ability to stimulatethe competence in Firmicutes, in particular Streptococcus, and morepreferably S. thermophilus strains.

According to the present invention, the length of the isolatedpolypeptide of the invention is less than 100 amino acids, preferablyless than 50 amino acids.

In a preferred embodiment, the isolated polypeptide of the presentinvention consists in the amino acids sequence SEQ ID No 1(LKTLKIFVLFSLLIAILPYFAGCL), or derivatives or fragments thereof, saidderivatives and fragments being capable of stimulating competence inFirmicutes, in particular Streptococcus, and more preferably S.thermophilus.

In another preferred embodiment, the isolated polypeptide of the presentinvention comprises, preferably consists in, a fragment of the aminoacids sequence SEQ ID No 1 capable of stimulating competence inFirmicutes, in particular Streptococcus, and more preferably S.thermophilus.

In still another embodiment, the isolated polypeptide of the presentinvention comprises, preferably consists in, the amino acids sequenceSEQ ID No 7 (IAILPYFAGCL) or derivatives or fragments thereof, saidderivatives and fragment being capable of stimulating competence inFirmicutes, in particular Streptococcus, and more preferably S.thermophilus.

As used herein, the term “fragment” refers to the products of thechemical, enzymatic, or physical breakdown of a polypeptide. Suchfragments may for example be obtained through enzymatic reaction, suchas degradation by protease and/or aminopeptidases.

Preferably, the length of such fragment is comprised between 3 and 17amino acids, preferably from 4 to 15 amino acids, and more preferablyfrom 6 to 12 amino acids.

In another preferred embodiment, the isolated polypeptide of the presentinvention further comprises an amino acid sequence corresponding to asignal peptide.

Said signal peptide allows the secretion of the polypeptide of theinvention in the extracellular medium when said polypeptide is expressedin a prokaryotic or an eukaryotic cell, preferably in a prokaryoticcell, such as in a bacteria from the Streptococcus genus, morepreferably in a Firmicutes, in particular Streptococcus, and morepreferably S. thermophilus bacteria.

Such signal peptides are well known from the skilled person.

As used herein, the term “derivatives”” refer to an amino acid sequencehaving a percentage of identity of at least 70% with the amino acidsequence SEQ ID No: 1, as an example at least 85% (i.e. 3 amino acidssubstitution), preferably of at least 90% (i.e. 2 amino acidssubstitution), and more preferably of at least 95% (i.e. 1 amino acidssubstitution).

As an example of derivative, one can cite the polypeptide of sequenceSEQ ID No 5 (MGKTLKIFVLFSLLIAILPYFAGCL), which is disclosed in theexamples.

As used herein, “percentage of identity” between two amino acidssequences, means the percentage of identical amino-acids, between thetwo sequences to be compared, obtained with the best alignment of saidsequences, this percentage being purely statistical and the differencesbetween these two sequences being randomly spread over the amino acidssequences. As used herein, “best alignment” or “optimal alignment”,means the alignment for which the determined percentage of identity (seebelow) is the highest. Sequences comparison between two amino acidssequences are usually realized by comparing these sequences that havebeen previously aligned according to the best alignment; this comparisonis realized on segments of comparison in order to identify and comparedthe local regions of similarity. The best sequences alignment to performcomparison can be realized, beside by a manual way, by using computersoftwares using such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA,TFASTA). The identity percentage between two sequences of amino acids isdetermined by comparing these two sequences optimally aligned, the aminoacids sequences being able to comprise additions or deletions in respectto the reference sequence in order to get the optimal alignment betweenthese two sequences. The percentage of identity is calculated bydetermining the number of identical position between these twosequences, and dividing this number by the total number of comparedpositions, and by multiplying the result obtained by 100 to get thepercentage of identity between these two sequences

As used herein an amino acid sequence having the ability to stimulatethe competence in Firmicutes, in particular Streptococcus, and morepreferably S. thermophilus strains can simply be identified by one ofskilled in the art in view of the following examples. As an example, theskilled person can screen for polypeptides derivated from SEQ ID No 1inducing competence in Firmicutes, in particular Streptococcus, and morepreferably S. thermophilus when present in the culture medium.

It will also be understood that natural amino acids may be replaced bychemically modified amino acids. Typically, such chemically modifiedamino acids enable to increase the polypeptide half life.

In a second aspect the present invention relates to an isolated nucleicacid encoding for the isolated polypeptide as described above.

Said nucleic acid corresponds to RNA or DNA, preferably to DNA.

According to a preferred embodiment, said isolated nucleic acidcomprises a nucleic acid sequence selected in the group comprisingTTGAAAACCCTGAAAATATTTGTACTATTTTCACTACTTATTGCTATCTTGCCTTATTTTGCAGGATGTCTTTTAA (SEQ ID No 2) andATGGGGAAAACCCTGAAAATATTTTGTACTATTTTCACTACTTATTGCTATCTTGCCTTATTTTGCAGGATGTCTTTAA (SEQ ID No 6).

In a third aspect, the present invention relates to a vector comprisingthe nucleic acid encoding for the isolated polypeptide of the inventionas described above operably linked to a gene expression sequence.

Said gene expression sequence directs the expression of said nucleicacid within a prokaryotic or an eukaryotic cell, preferably within aprokaryotic cell, and more preferably within Firmicutes, in particularStreptococcus, and more preferably S. thermophilus bacteria. The “geneexpression sequence” is any regulatory nucleotide sequence, such as apromoter sequence or promoter-enhancer combination, which facilitatesthe efficient transcription and translation of the nucleic acid to whichit is operatively linked. The gene expression sequence may be aconstitutive or inducible promoter.

Such promoters are well known in the art. Promoters for use in theinvention are preferably strong promoters, i.e. on induction in therelevant cell yield high levels of transcription of the downstream gene.Examples of strong inducible promoters include by examples promoters,from bacteria, involved in the production of bacteriocins such as thestb promoter.

In general, the gene expression sequence shall include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription and translation, respectively. The geneexpression sequences optionally include enhancer sequences or upstreamactivator sequences as desired.

As used herein, the nucleic acid sequence encoding the polypeptide ofthe invention and the gene expression sequence are said to be “operablylinked” when they are covalently linked in such a way as to place theexpression or transcription and/or translation of the polypeptide of theinvention coding sequence under the influence or control of the geneexpression sequence. Two DNA sequences are said to be operably linked ifinduction of a promoter in the 5′ gene expression sequence results inthe transcription of the polypeptide of the invention and if the natureof the linkage between the two DNA sequences does not result in theintroduction of a frame-shift mutation, interfere with the ability ofthe promoter region to direct the transcription of the polypeptide ofthe invention, or interfere with the ability of the corresponding RNAtranscript to be translated into a protein. Thus, a gene expressionsequence would be operably linked to a nucleic acid sequence coding forthe polypeptide of the invention if the gene expression sequence wascapable of effecting transcription of that nucleic acid sequence suchthat the resulting transcript is translated into the desiredpolypeptide.

Preferred vectors are plasmid vectors, which have been extensivelydescribed in the art and are well known to those of skilled in the art.See e.g., SAMBROOK et al., “Molecular Cloning: A Laboratory Manual,”Second Edition, Cold Spring Harbor Laboratory Press, 1989.

The vector of the invention can include a selectable marker that isactive in bacteria.

In a forth aspect, the present invention relates to a host cellgenetically engineered with the vector described previously.

As used herein, the term “host cell genetically engineered” relates tohost cells which have been transformed with the vector describedpreviously.

Said host cell is preferably a bacterial cell, such as a bacteriabelonging to the Firmicutes phylum, preferably the Streptococcous genus,and more preferably a Streptococcus thermophilus bacteria.

The introduction of the vector described previously into the host cellcan be effected by method well known from one of skilled in the art suchas calcium phosphate transfection or electroporation.

In a fifth embodiment, the present invention relates to a culture mediumcomprising an effective amount of the isolated polypeptide as describedabove and nutrients that allow the growing of Firmicutes, in particularStreptococcus, and more preferably S. thermophilus.

According to the present invention, an “effective amount” of saidisolated polypeptide is one which is sufficient to achieve a desiredbiological effect, in this case stimulating competence in Firmicutes, inparticular Streptococcus, and more preferably S. thermophilus. As anexample, said effective amount is comprised between 0.1 ng/ml and 1mg/ml, preferably between 0.5 ng/ml and 1 mg/ml, and more preferablybetween 1 ng/ml and 100 ng/ml.

Nutrients of the culture medium are well known from the skilled personand include, as for example lactose, sodium acetate, ascorbic acid,potassium phosphate. Preferably, the medium culture does not contain anynutritional peptides, such as those provided by neopeptone or yeastextract.

There are numerous causes of peptide instability or degradation,including hydrolysis and denaturation. This result may entail diminutionof the induction of the competence in Firmicutes, in particularStreptococcus, and more preferably S. thermophilus. Stabilizers may beadded to lessen or prevent such problems.

According to a specific embodiment, the culture medium of the inventionfurther comprises at least one stabilizer.

Stabilizers include cyclodextrine and derivatives thereof (see U.S. Pat.No. 5,730,969). Suitable preservatives such as sucrose, mannitol,sorbitol, trehalose, dextran and glycerin can also be added to stabilizethe final formulation. Polyols may stabilize a peptide, and arewater-miscible or water-soluble. Suitable polyols may be polyhydroxyalcohols, monosaccharides and disaccharides including mannitol,glycerol, ethylene glycol, propylene glycol, trimethyl glycol, vinylpyrrolidone, glucose, fructose, arabinose, mannose, maltose, sucrose,and polymers thereof. Various excipients may also stabilize peptides,including serum albumin, amino acids, heparin, fatty acids andphospholipids.

In a sixth aspect, the present invention relates to the use of apolypeptide as described above, a nucleic acid as described previously,a vector comprising such a nucleic acid, a host cell as definedpreviously, or a culture medium as described previously for stimulatingcompetence in Firmicutes, in particular Streptococcus, and morepreferably S. thermophilus.

In a seventh aspect, the present invention relates to a method ofproducing transformation competent Firmicutes, in particularStreptococcus, and more preferably S. thermophilus bacteria comprisingthe step (i) of contacting said Firmicutes, in particular Streptococcus,and more preferably S. thermophilus bacteria with an effective amount ofan isolated polypeptide as defined previously for obtaining saidtransformation competent Firmicutes, in particular Streptococcus, andmore preferably S. thermophilus bacteria.

Preferably, the method of the invention is performed in a culture mediumas described previously.

In a eight aspect, the present invention relates to a method ofproducing transformation competent Firmicutes, in particularStreptococcus, and more preferably S. thermophilus bacteria comprisingthe step (i) of culturing said Firmicutes, in particular Streptococcus,and more preferably S. thermophilus bacteria in a peptide free mediumallowing the growth of said bacteria to an OD₆₀₀ comprised between 1.5and 2.5 preferably about 2 and (i′) diluting said culture to an OD₆₀₀comprised between 0.01 and 0.1, preferably about 0.05.

Preferably, the present invention relates to a method of producingtransformation competent Firmicutes, in particular Streptococcus, andmore preferably S. thermophilus bacteria comprising the step (i) ofculturing said Firmicutes, in particular Streptococcus, and morepreferably S. thermophilus bacteria in M17 lactose (10 g/l) for 8 hoursand (i′) diluting said culture 50 fold in a peptide free medium. Thediluted culture is then incubated at 4° C. for 10 hours and furtherincubated at 42° C. for 6 hours allowing the growth of said bacteria toan OD600 comprised between 1.5 and 2.5 preferably about 2, and (i″)diluting said culture in a peptide free medium to an OD600 comprisedbetween 0.01 and 0.1, preferably about 0.05.

For example, said peptide free medium comprises:

g/mol g/l Lactose/Buffer lactose 360.32 10 g Na acetate 82.03 1 gammonium citrate 243.2 0.6 g KH2PO4 136.09 3 g K2HPO4 174.18 2.5 g urea60.06 0.240 g Vitamines  1 Ascorbic acid L+ 176.13 0.5 g  2 pyridoxamin241.1 5 mg  3 nicotinic acid 123.1 1 mg  4 riboflavin 376.4 1 mg  5panthothenic acid 238.3 1 mg  6 thiamin 337.3 1 mg  7 pyridoxin 205.6 2mg  8 aminobenzoic acid 137.1 10 mg  9 biotin 244.3 10 mg 10 folic ac441.4 1 mg 11 B12 1355.4 1 mg 12 orotic ac 156.1 5 mg 13 thymidine 242.25 mg 14 inosin 268.23 5 mg 15 DL 6,8 thioctic ac 206.3 2.5 mg MetalsMgCL2—6H2O 203.3 0.200 g CaCL2—2H2O 147.02 0.050 g FeCl2 198.81 0.005 gZnS04 287.54 0.005 g CuSO4 249.7 0.000 g CoCl2 237.9 0.003 g MnS04 1690.028 g Amino Acid L aspartic acid 133.1 0.455 g L glutamic acid 147.130.398 g L asparagine 132.1 0.350 g L glutamine 146.1 0.390 g L histidine155.16 0.150 g L arginine 174.2 0.350 g L lysine 146.2 0.440 g L serine105.1 0.225 g L threonine 119.1 0.175 g glycine 75.07 0.175 g L alanine89.1 0.240 g L isoleucine 131.18 0.210 g L leucine 131.18 0.475 g Lvaline 117.1 0.325 g L tryptophan 204.2 0.050 g L méthionine 149.2 0.125g L proline 115.13 0.675 g L phenylalanine 165.19 0.275 g L tyrosine181.2 0.290 g L cystéine 121.16 0.250 g Nucleic acids adenine 135.10.010 g uracil 112.09 0.010 g xanthine 174.1 0.010 g guanine 187.6 0.010g

The medium is adjusted to pH 6.6 with HCl, completed to 1 L with waterand filtered on GV filter.

Advantageously, said Firmicutes, in particular Streptococcus, and morepreferably S. thermophilus bacteria is selected in the group comprisingS. thermophilus LMD-9, S. thermophilus CNRZ1066 and S. thermophilusLMG18311, preferably S. thermophilus LMD-9.

According to a preferred embodiment, said methods are for producing amutant bacteria and said method further comprises the step (ii)contacting said transformation competent Firmicutes, in particularStreptococcus, and more preferably S. thermophilus bacteria withhomologous DNA under conditions to allow transformation of said bacteriawith said homologous DNA and insertion of the homologous DNA in thechromosome of Firmicutes, in particular Streptococcus, and morepreferably S. thermophilus by homologous recombination.

Said homologous DNA can be linear DNA (PCR fragment), plasmid DNA orchromosomal DNA.

Advantageously, said contacting step (ii) is realised simultaneouslywith the step (i′) of diluting the culture of the method according tothe eighth aspect. In that case, the transformation happened essentiallyduring the 100 minutes following said contacting, more specificallyessentially during the first 60 minutes.

Advantageously, said methods may comprise the steps (iii) of selectingand/or amplifying the mutant bacteria thus generated.

In a ninth aspect, the present invention concerns a method of screeninga compound, preferably a polypeptide, capable of stimulating competencein Firmicutes, in particular Streptococcus, and more preferably S.thermophilus using the oligopeptide binding protein AmiA3 target andcomprising the steps of contacting said compound with AmiA3, verifyingthe binding between AmiA3 and said compound, and selecting compound thatbind to AmiA3 as a compound capable of stimulating competence inFirmicutes, in particular Streptococcus, and more preferably S.thermophilus.

Methods for verifying the binding between AmiA3 and said compound arewell known by the skilled person.

In a tenth aspect, the present invention concerns a method foridentifying a compound stimulating competence in Firmicutes, inparticular Streptococcus, and more preferably S. thermophilus comprisingthe steps of:

-   -   i) contacting, with said compound, a host cell (preferably a        bacteria) transformed with a nucleic acid comprising a nucleic        acid sequence coding for a reporter protein (preferably GFP,        béta Galactosidase, etc.) under the control of all or part of a        promoter preceded by the inverted repeat sequence recognized by        the P1cR like regulator (Ster0316 in strain LMD9, str0270 in        strain CNRZ1066, stu0270 in strain LMG18311)        ATAGTGACATATATGTCTCTAT (SEQ ID No 3) or GTGGTGACATAAATGTCACTAT        (SEQ ID No 4);    -   ii) selecting the compound that stimulates the expression of        said reporter protein.

Methods for testing the stimulation of Firmicutes, in particularStreptococcus, and more preferably S. thermophilus competence are wellknown from the skilled person. Examples of such methods are disclosed inthe examples.

In a preferred embodiment, the method of the invention can furthercomprises the steps of:

-   -   iii) contacting said compound with Firmicutes, in particular        Streptococcus, and more preferably S. thermophilus in the        presence of DNA (plasmids, chromosomal DNA, etc.); and    -   iv) selecting the compounds that effectively enhance competence.

In the following, the invention is described in more detail withreference to amino acid sequences, nucleic acid sequences and theexamples. Yet, no limitation of the invention is intended by the detailsof the examples. Rather, the invention pertains to any embodiment whichcomprises details which are not explicitly mentioned in the examplesherein, but which the skilled person finds without undue effort.

EXAMPLES 1) The Anti Transporter Controls the Synthesis of SeveralProteins Essential for Natural Transformation in Streptococci

In order to find physiological functions controlled by signalingpeptides that are internalized by the Ami transporter, we compared theproteome of the wild type LMD-9 strain and its isogenic mutant deletedfor the ami operon, LMD-9 ΔamiCDE (TIL883; IBRAHIM et al., J.Bacteriol., vol. 189, p: 8844-8854, 2007).

In order to by-pass the nutritional function of the Ami transporter,cells were gown in CDM, a free-peptide chemically defined medium (CDM),containing only amino acids as nitrogen source, as described by Letort &Juillard, Development of a minimal chemically-defined medium for theexponential growth of Streptococcus thermophilus, J. Appl. Microbiol.,vol. 91, p. 1023-1029, 2001. Optical density at 600 nm (OD₆₀₀) of thecultures was measured using a spectrophotometer UVIKON 931 (KONTRON).

Proteins were prepared from cells grown in CDM and harvested at OD₆₀₀0.7 in two independent cultures for each strain. Bacteria weremechanically disrupted and the supernantants were ultracentrifuged at220,000 g for 30 min at 4° C. to enrich the ‘cell envelope pellets’ incell-envelope proteins. Finally the pellet were resuspended indisruption buffer and sonicated for 15 min at 4° C. in an ultrasonicbath. The cell-envelope pellet fractions (10 gag) were separated by 1Delectrophoresis. Each 1D electrophoresis lane was cut into 26 pieces ofgel (2 mm width). In-gel digestion of the proteins was performed withthe Progest system (Genomic Solution) according to the followingprotocol.

Gel pieces were washed firstly, in two successive baths of (i) 10%acetic acid 40% ethanol and (ii) 100% acetonitrile (ACN) and secondly,in two successive baths of (i) 25 mM NH₄CO₃ and (ii) 100% ACN. Gelpieces were further incubated in 10 mM DTT in 25 mM NH₄CO₃, 30 mM at 55°C. and in 50 mM iodoacetamide in 25 mM NH₄CO₃, 45 mM at room temperaturefor cysteine reduction and alkylation, respectively. Digestion wassubsequently performed for 6 h at 37° C. with 125 ng of modified trypsin(PROMEGA) dissolved in 20% methanol and 20 mM NH₄CO₃, per gel piece. Thepeptides were extracted successively with (i) 0.5% trifluoroacetic acid(TFA) 50% ACN and (ii) with 100% ACN. The resulting peptide extractswere dried in a vacuum centrifuge and suspended in 25 μl of 0.08% TFA,and 2% ACN.

Fractions enriched in cell envelope proteins were then analyzed by alabel-free comparative proteomic approach combining 1D electrophoresiswith LC-MS/MS analysis.

LC-MS/MS analysis was performed on Ultimate 3000 LC system (DIONEX)connected to LTQ Orbitrap mass spectrometer (THERMO FISHER) bynanoelectrospray ion source. Tryptic peptide mixtures (4 μl) were loadedat flow rate 20 μl min⁻¹ onto precolumn Pepmap C18 (0.3×5 mm, 100 Å, 5μm; DIONEX). After 4 mM, the precolumn was connected with the separatingnanocolumn Pepmap C18 (0.075×15 cm, 100 Å, 3 μm) and the linear gradientwas started from 2 to 36% of buffer B (0.1% formic acid, 80%acetonitrile) in buffer A (0.1% formic acid, 2% acetonitrile) at 300 nlmin⁻¹ over 50 min. Ionization was performed on liquid junction with aspray voltage of 1.3 kV applied to non-coated capillary probe (PicoTipEMITER 10 μm tip ID; NEW OBJECTIVE). Peptides ions were automaticallyanalyzed by the data dependent method as follows: full MS scan (m/z300-1600) on Orbitrap analyser and MS/MS on the 4 most abundantprecursor on the LTQ linear ion trap. In this study only +2 and +3charged peptides were subjected to MS/MS experiments with an exclusionwindow of 1.5 min, with classical peptides fragmentation parameters:Qz=0.22, activation time=50 ms, collision energy=35%.

The raw data produced on LTQ-Orbitrap mass spectrometer were firstconverted in mzXML file with ReADW (http://sashimi.sourceforge.net) andin a second step, protein identification was performed with X!Tandemsoftware (X!Tandem tornado 2008.02.01.3, http://www.thegpm.org) againsta protein database of S. thermophilus LMD-9 (GenBank: CP000419.1),associated to a proteomic contaminant database. The X!Tandem searchparameters were: trypsin specificity with one missed cleavage, fixedalkylation of cysteine and variable oxydation of methionine. The masstolerance was fixed to 10 ppm for precursor ions and 0.5 Da for fragmentions. For all proteins identified with a protein E-value <0.01 in thefirst step, we searched for additional peptides to reinforceidentification using similar parameters except that semi-trypticpeptides and protein N-terminal acetylations were accepted. All peptidesidentified with an E-value <0.1 were conserved. All results for eachpiece of gel were merged with an home-made program written in java byBenoit Valot at the PAPPSO platform (http://moulon.inra.fr/PAPPSO). Thefinal search results were filtered using a multiple threshold filterapplied at the protein level and consisting of the following criteria:protein E-value <10⁻⁸ identified with a minimum of two differentpeptides sequences, detected in at least one piece of gel, with anpeptide E-value <0.05.

We focused our attention on proteins that were detected in the extractsprepared from strain LIVID-9 and that completely disappeared in theextracts prepared from strain TIL883. The identified proteins aredisclosed in the Table I.

For each protein detected, we calculated an abundance factor defined asthe total number of spectra detected per protein in each gel lanenormalized by the theoretical number of peptides having a mass rangingbetween 800 and 2500 Da. Proteins that were detected in the tworepetitions performed for strain LMD-9 and absent in the two repetitionsperformed for strain TIL883 (abundance factor=0) were taken intoaccount.

TABLE I Abundance factor^(a) GenBank MW Protein identification LMD-9TIL883 Proteins essential for natural transformation^(b) STER1521 24400DNA uptake protein or related DNA- 0.63-0.50 0-0 binding protein, ComEASTER0922 31100 Predicted Rossmann fold nucleotide- 1.12-1.12 0-0 bindingprotein involved in DNA uptake, DprA STER1821 14700 Single-strandedDNA-binding protein, 0.50-0.83 0-0 SsbB STER1840 11800 Competenceprotein ComGC   1-1.75 0-0 STER1841 33800 Type II secretory pathway/0.64-0.82 0-0 competence component, ComGB STER1842 35300 Type IIsecretory pathway/ 0.95-0.79 0-0 competence component, ATPase, ComGASTER0189 20100 ComX 0.57-0.14 0-0 Proteins induced by the competencestate but not essential for transformation^(b) STER0057 30100 Surfaceantigen, CbpD 0.75-0.75 0-0 STER1430 25700 DNA repair protein, RadC0.25-0.33 0-0 Subunits of the Ami oligopeptide transport systemSTER_1407 34600 ABC-type dipeptide/oligopeptide/ 2.17-2.33 0-0 nickeltransport system, permease component, AmiD STER_1408 55500 ABC-typedipeptide/oligopeptide/ 1.41-1.12 0-0 nickel transport system, permeasecomponent, AmiC STER_1406 39800 ABC-type dipeptide/oligopeptide/1.28-1.  0-0 nickel transport system, ATPase component, AmiE Otherproteins STER_1356 50500 Radical SAM superfamily enzyme 1.55-1.65 0-0STER_1652 50300 lactococcin A ABC transporter 0.60-1.00 0-0 permeaseprotein, PcsB STER_0329 32200 Urease accessory protein UreH 0.57-0.570-0 STER_0123 18500 Predicted RNA-binding protein 0.60-0.40 0-0containing a PIN domain STER_1296 45700 Permease of the majorfacilitator 0.57-0.29 0-0 superfamily STER_0331 29100 ABC-type cobalttransport system, 0.23-0.46 0-0 permease component CbiQ or relatedtransporter STER_1779 12200 Thioredoxin domain containing protein0.33-0.33 0-0 STER_1834 43200 Acetate kinase 0.26-0.32 0-0 ^(a)Theabundance factor is the ratio between the total number of spectraobtained during the protein identification process on the theoreticalnumber of peptides ranging between 800 and 2500 Da. Two repetitions wereperformed for each strain leading to two values. ^(b)These categories ofproteins have been defined from the results obtained with theorthologues of these proteins in S. pneumoniae.

The results show that in addition to the subunits C, D and E of the Amitransporter, which were absent from the mutant, as expected, seventeenproteins fulfilled this criterion (Table I). Eight were encoded by genesthe orthologues of which were identified as late CSP-induced genes in S.pneumoniae. Among them, six have also been identified as essential fornatural transformation (Peterson et al., Identification of competencepheromone responsive genes in Streptococcus pneumoniae by use of DNAmicroarrays. Mol. Microbiol. 51:1051-1070, 2004). The ComX proteininvolved in the regulation of the competence state was also detected instrain LMD-9 but not in strain TIL883.

These results suggest that the Ami transporter is involved in theregulatory pathway that controls the induction of the competence statein S. thermophilus and also that natural transformation can be turned onin CDM during the exponential growth phase.

2) The S. thermophilus LMD-9 is Naturally Transformable in CDM.

To check the hypothesis formulated on the basis of our proteomicresults, we first tested the natural transformability of S. thermophilususing the pG⁺host9 plasmid.

An overnight culture of strain LMD-9 grown in CDM at 42° C. was dilutedin CDM to OD₆₀₀ 0.05. 2 ml of this diluted culture were distributed in2-ml tubes and incubated in a water bath at 42° C. Once each hour forfour hours, a sample was used to measure the OD₆₀₀ and 100 μl were mixedwith 1 μg of plasmid DNA. Cells with DNA were incubated for 2 hours at28° C. before being plated on M17 medium (DIFCO) supplemented with 10 gliter⁻¹ lactose (M17lac) with erythromycin (5 μg for S. thermophilus).

The FIG. 1 shows the development of competence during growth of strainLMD-9 in CDM using pG⁺host9 plasmid as transformant DNA. Optical density(OD₆₀₀) (x, dashed line) was used to measure cell numbers and count ofcells resistant to erythromycin (Ery resistant cells) (♦, plain line)was used to assess competence. 1 μg of plasmid DNA was mixed with 100 μlof cells. The mean of three independent experiments are presented anderror bars indicate standard deviation.

We obtained erythromycin resistant cells (transformants) but only insamples harvested one hour after dilution (FIG. 1). We observed a meanof 1.2×10⁶ transformants per ml (standard error, ±7.2×10⁵ transformantsper ml; n=3). The presence of the plasmid pG⁺host9 in these bacteria waschecked on several colonies by PCR.

This result indicates that bacteria were able to take up the plasmid butonly at a specific growth stage corresponding to the beginning of theexponential phase (OD₆₀₀ 0.2-0.3).

To confirm the transformability of S. thermophilus, to demonstrate itsability to take up linear DNA (PCR fragment or chromosomal DNA) and toincorporate it by homologous recombination in its chromosome and also toassess the kinetics of the transformation rate more precisely, we neededchromosomal DNA containing an antibiotic resistant marker.

For that purpose, we constructed strain TIL1192 (LMD-9 feoB::erm)containing an erythromycin (erm) resistant cassette introduced into thechromosome of strain LMD-9 at the feo locus. Integration of a PCRfragment by homologous recombination in the chromosome of strain LMD-9was demonstrated in the framework of this construction.

Chromosomal DNA of strain TIL1192 was further used as donor DNA to studythe timing of the triggering of the competence state during growth inCDM.

An overnight culture of strain LMD-9 grown in CDM at 42° C. was dilutedin CDM to OD₆₀₀ 0.05. 2 ml of this diluted culture were distributed in2-ml tubes and incubated in a water bath at 42° C. Once twenty minutesfor two hours, a sample was used to measure the OD₆₀₀ and 100 μl weremixed with 1 μg of chromosomal DNA. Cells with DNA were incubated for 2hours at 42° C. before being plated on M17 medium (DIFCO) supplementedwith 10 g liter⁻¹ lactose (M17lac) with erythromycin (5 μg ml⁻¹ orkanamycin 1000 μg/ml⁻¹ for S. thermophilus).

The FIG. 2 shows the development of competence during growth of strainLMD-9 in CDM using chromosomal DNA of strain TIL1192 as transformantDNA. Optical density (OD₆₀₀) (x, dashed line) was used to measure cellnumber and count of cells resistant to erythromycin (Ery resistantcells) (♦, plained line) was used to assess competence. 1 μg ofchromosomal DNA was mixed with 100 μl of cells. The mean of fourindependent experiments are presented and error bars indicate standarddeviation.

The kinetics of transformation obtained from four independentexperiments (FIG. 2) confirmed that natural competence is a shorttransitory state. Transformability rose sharply 20 min after dilution(OD₆₀₀ 0.06), reached an optimum one hour after dilution (OD₆₀₀0.17˜0.2) and then rapidly declined. One hundred min after dilution(OD₆₀₀ 0.4˜0.5), cells were no longer transformable. At the optimum, theaverage transformation rate was 3.8 10⁻⁶ (standard error, ±4.6 10⁻⁷;n=4).

We also tested the transformability of strain TIL883 (ΔamiCDE) withchromosomal DNA of TIL1192 under the same conditions. No transformantswere obtained during the growth of this strain in CDM.

In order to confirm that antibiotic resistant clones obtained from theprevious experiments were the result of a natural transformationinvolving ComEC and most probably a transformasome complex similar tothe one described in S. pneumoniae, we constructed strain TIL1195 (LMD-9comEC::erm). ComEC is one of the proteins of the DNA uptake machineryessential for natural transformation in S. pneumoniae and B. subtilis.

We also constructed TIL1193 (LMD-9 feo::aphA3) as a chromosomal DNAsource with a different antibiotic resistance than erythromycin. Wechecked that natural transformation of strain LMD-9 with chromosomal DNAof strain TIL1193 gave a similar transformation rate as with chromosomalDNA of strain TIL1192 (data not shown). Finally, we tried to naturallytransform strain TIL1195 with chromosomal DNA of strain TIL1193. Samplesof cells of TIL1195 grown in CDM were harvested every 30 min for 2 hoursand tested for transformation.

We obtained no kanamycin-resistant clones.

In order to confirm that ComX is essential for natural transformation inS. thermophilus, we constructed strain TIL1196 (LMD-9 comX::erm).Samples of strain' cells TIL1196 grown in CDM were harvested every 30min for 2 hours and tested for transformation with chromosomal DNA ofstrain TIL1193.

As expected, we obtained no kanamycin-resistant clones during thiskinetic.

Finally, we have found a natural condition of growth that turns on thetransformability of strain LMD-9. Using plasmid or chromosomal DNA asdonor DNA, we showed that cells were transformable in CDM, during anarrow window, the optimum being one hour after the dilution of anovernight culture in CDM. The rates obtained made it possible to easilyconstruct deletion mutants using PCR fragments and were much higher withplasmid DNA than with chromosomal DNA. This difference can be explainedby two factors. First, the higher number of molecules of plasmidcompared to the number of molecules of chromosome present in 1 μg DNA(around 450 fold more). Second, the pG+host plasmids generate linearplasmid multimers in Lactococcus lactic (Magnin et al., Efficientinsertional mutagenesis in lactococci and other gram-positive bacteria.J. Bacteriol. 178:931-935, 1996). Such is probably also the case in S.thermophilus.

3) The Ami Transporter Indirectly Controls the Transcription of GenesNecessary for the Development of Competence in Streptococci.

Among the proteins detected in strain LMD-9 and not in strain TIL883,three were chosen for a transcriptional study of the correspondinggenes. These proteins were ComGA that is involved in the pore assembly,DprA, a recombination mediator protein that conveys incoming ssDNA tothe recombinase RecA and the sigma factor ComX. Although the abundancefactor of RecA did not reach zero in the strain TIL883 but decreased bya factor 4 (data not shown), we chose to follow its encoding genebecause RecA is essential for transformation in many Gram-positivetransformable species.

The FIG. 3 shows the relative expression levels of comX, recA, dprA andcomGA between S. thermophilus LMD-9 and strain TIL883 (LMD9 ΔamiCDE) orstrain TIL1196 (LMD-9 comX::erm). Relative expression levels werecomputed using the comparative critical threshold method (2^(−ΔΔC)T) asdescribed by Livak and Schmittgen (Analysis of relative gene expressiondata using real-time quantitative PCR and the 2(−ΔΔ C(T)) Method.Methods, 25:402-408, 2001). Data are expressed as means from threeindependent experiments and were significant according to an analysis ofvariance (P<0.05).

As shown in FIG. 3, the level of expression of comGA and dprA of strainLMD-9 was higher than that of strain TIL883. To a lesser extent but withsignificant values (P<0.05), we obtained similar results with comX andrecA that were 5-fold and 4-fold more highly expressed in strain LMD-9,respectively.

To confirm that the transcription of genes dprA, comGA and recA is underthe control of ComX, we compared the expression of these genes in strainLMD-9 and strain TIL1196 (LMD9 comX::erm). As expected, thetranscription of the three genes was significantly higher in strainLMD-9 than in strain TIL1196 (FIG. 3) confirming that theirtranscription is positively controlled by ComX.

4) The Oligopeptide-Binding Protein AmiA3 Plays the Major Role in theControl of Competence.

The genome of strain LMD-9 displays only two genes encodingoligopeptide-binding proteins, amiA1 (ster_(—)1409), the first gene ofthe ami operon (ster_(—)1408 to ster_(—)1405) and amiA3 (ster_(—)1411)that is flanked by two transposase encoding genes.

We constructed three strains, TIL1197 (amiA3::erm), TIL1198 (ΔamiA1) andTIL1199 (amiA3::erm ΔamiA1) corresponding to insertional mutagenesis ofamiA3, deletion of amiA1 and a combination of both mutations,respectively.

The transformability of the three strains was tested using chromosomalDNA of strain TIL1193 and compared to that of strain LMD-9 at itsoptimum of competence, i.e. one hour after the dilution of the cells inCDM.

As expected, no kanamycin resistant cells were obtained aftertransformation of strain TIL1199. However, the percentage of thecompetence rate of strains TIL1197 (amiA3::erm) and TIL1198 (ΔamiA1)compared to that of strain LMD-9 were 1% (standard error±2) and 48%(standard error±4), respectively.

This result suggests that AmiA3 is more important in the triggering ofthe competence than AmiA1.

As our experiments were performed in a medium without peptides, wehypothesize that in S. thermophilus, the Ami3 oligopeptide-bindingprotein imports a peptide involved with a transcriptional regulator inthe control of the expression of comX. This peptide could be a specificpheromone or a peptide resulting from the degradation of secretedproteins or proteins released by lysis of bacteria.

5) The Growth Medium Composition Influences the Competence State of S.thermophilus.

Kinetics of competence rate were performed with LMD-9 cells grown inM17lac and with pG⁺host9 plasmid DNA or chromosomal DNA of strainTIL1192.

With both types of donor DNA, no erythromycin resistant transformantswere obtained with cells harvested every 30 min for 2 hours.

We then compared the expression of comGA, dprA, recA and comX from RNAextracted from LMD-9 cells grown in CDM and M17lac medium and harvestedat OD₆₀₀ 0.2.

We observed that these genes were respectively, 1492, 2246, 24 and 236more highly expressed in CDM than in M17lac which was consistent withthe absence of transformants during growth in M17lac.

6) Strains CNRZ1066 and LMG18311 are not efficiently transformable inCDM.

We tested natural transformability of the two other S. thermophilusstrains, CNRZ1066 and LMG18311.

For this purpose, we used the plasmid pG+host9 as donor DNA and cellswere harvested every 30 min for 2 hours.

We obtained no erythromycin resistant clones with strain CNRZ1066 and afew erythromycin resistant clones with strain LMG18311, one hour afterdilution. However, we obtained 2 10⁴ less transformants with strainLMG18311 than with strain LMD-9. We also used chromosomal DNA of strainTIL1195 (LMD-9 comEC::erm) as donor DNA because surrounding regions ofgene comEC are highly conserved between strains LMD-9, CNRZ1066 andLMG18311 (more than 98% identity over 5 kb upstream and downstreamcomEC). Under this condition, we obtained no erythromycin resistantclones with both strains.

7) Identification of a Competence Stimulating Peptide in S. thermophilus

During the proteomic approach described in paragraph 1, we noticed thatthe synthesis of a transcriptional regulator annotated PlcR, ster0316,decreased in the Ami mutant compared to the wild type strain. Theseregulators are known to be regulated by peptides that are secreted andimported back by Opp. As we suspected that the triggering of thecompetence state in S. thermophilus is controlled by a secreted peptidefurther imported by Opp also called Ami in this species, we thought thatthis regulator could be involved in the mechanism controlling thetriggering of competence in S. thermophilus. We deleted ster0316 andreplaced it by an erythromycin resistance cassette. The transformabilityof the mutant was assessed for three hours every thirty minutes usingchromosomal DNA of strain TIL1193 (feo::aphA3). Three independentexperiments were performed and no kanamycin resistant transformants wereobtained indicating that ster0316 is involved in the triggering ofcompetence in S. thermophilus.

A small CDS, papR-like, is located downstream of gene ster0316 and isnot annotated in Genbank. As the activity of PlcR regulators iscontrolled by peptides, we suspected that the peptide encoded by thissmall papR-like CDS could be involved in the control of the activity ofSter0316. papR-like was deleted and replaced by a spectinomycinresistance cassette leading to the construction of a papR-like::specmutant. The transformability of the mutant was assessed for three hoursevery thirty minutes using plasmid DNA (pGhost9). Four independentexperiments were performed and no erythromycin resistant transformantswere obtained indicating that papR-like is most probably involved in thetriggering of competence in S. thermophilus probably through the controlof the activity of Ster0316.

In order to check that the absence of transformability of thepapR-like::spec mutant was the result of the absence of the papR-likegene and not the result of a polar effect of the presence of thespectinomycin resistant cassette on upstream or downstream genes ofpapR-like, we cloned the papR-like gene in a plasmid in order to expressit under the control of a strong constitutive promoter and introducedthis plasmid by electroporation in the papR-like::spec mutant. Thetransformability of the mutant was assessed one hour after the dilutionof the preculture in CDM using plasmid DNA (pGhost9::kana). We obtainedkanamycin resistant transformants indicating that the transformabilityof the papR-like::spec mutant was restored by the presence of papR andthat the absence of transformality of the papR-like::spec mutant was theresult of the absence of the papR-like gene.

8) Protocol of Induction of Competence and Transformation of S.thermophilus Comprising Medium CDM and CSP (Competence StimulatingPeptide).

As used herein, CSP refers to Pap-R like peptide or a fragment thereof.

S. thermophilus cells are grown overnight at 42° C. in CDM. The cultureis then diluted in CDM at an OD₆₀₀ 0.05. Sixty minutes after dilution,the competence stimulating peptide (CSP), which is the mature peptidecomprising SEQ ID No 1 or fragments or derivatives of SEQ ID No 1 thatallow to induce competence in S. thermophilus, is added to the cultureat a final concentration of 1 μM. Ten minutes later, 100 μl of theculture containing the CSP is mixed with 1 ng of DNA and incubated for 2hours at 28° C. when mixed with a thermosensitive replicative plasmidDNA or 1 hour at 42° C. when mixed with chromosomal DNA or PCRfragments, before being serially diluted and spread on M17lac plateswith the appropriate antibiotic.

9) Overexpression of the papR-like Gene Induces the Transformability ofStrain LMG18311

Strain S. thermophilus LMG18311 is naturally poorly transformable. Weintroduced in this strain and by electroporation, the plasmid comprisingthe sequence SEQ ID No 6 coding for the polypeptide derivatives havingthe sequence SEQ ID No 5, plasmid that allows the overexpression of thepapR-like gene and that is described in paragraph 7. Thetransformability of the mutant was assessed one hour after the dilutionof the culture of this mutant in CDM using plasmid DNA (pGhost9::kana).100 μl of the culture was mixed with 1 μg of DNA. Cells were furtherincubated 2 hours at 30° C. before been spread on M17lac platescontaining the appropriate antibiotic. The LMG18311 wild type strain wasused as a control. We obtained no kanamycin resistant transformants withthe control and many (>1000) with the mutant overexpressing thepapR-like gene. This result indicates that the overexpression of thepapR-like gene is able to stimulate the transformability of a poorlytransformable strain.

10) Transformation of S. thermophilus by a Plasmid that Allows theOverexpression of the papR-like Gene

S. thermophilus cells are electroporated with a replicative plasmidwhere the papR-like gene is under the control of a strong constitutivepromoter. Cells containing this plasmid are grown overnight at 42° C. inCDM. The culture is then diluted in CDM at an OD₆₀₀ 0.05. One hour afterdilution, 100 μl of the culture is mixed with 1 μg of DNA and incubatedfor 2 hours at 28° C. when mixed with a thermosensitive replicativeplasmid DNA or 1 hour at 42° C. when mixed with chromosomal DNA or PCRfragments, before being serially diluted and spread on M17lac plateswith the appropriate antibiotic. The transformants can further be easilycured of the replicative plasmid by growth in the absence of antibiotic.

11) Identification of Different Fragments of SEQ ID No 1

SEQ ID No 1 is the precursor of shorter peptides that are secreted.These shorter peptides, corresponding to fragments of SEQ ID No 1, areactive peptides that trigger the competence state in S. thermophilus.

For identifying the sequence of these shorter active peptides, theinventors have used two strains, one that is deleted for the papR-likegene (referred as “deleted strain” thereafter) and one thatoverexpresses the papR-like gene and that is unable to import peptidesbecause deleted for the genes encoding the oligopeptide transporter Ami(referred as “overproducing strain” thereafter). In the overproducingstrain, the sequence of the peptide that is overproduced is SEQ ID No 5.

Using LC-MS/MS analysis, the inventors have searched for peptide massescorresponding to fragments of SEQ ID No 1 in the culture supernatant ofthe overproducing strain that were absent in that of the deleted strain.Culture supernatants were treated in order to be enriched in peptideswith the following method:

-   -   (1) ultrafiltration (10 kDa cut-off),    -   (2) injection of the ultrafiltrate (<10 kDa) on Sep-pack C18        with a 30% acetonitrile washing step and a 40% acetonitrile        elution step. Eluted fractions were further dried and        resuspended with 0.1% trifluoroacetic acid 2% acetonitrile.

Three masses corresponding to masses of five different fragments of SEQID No 1 (present in the overproducing strain and absent in the deletedstrain) have been identified, with different retention times (Rt) duringthe HPLC runs (A-natural peptides, below). The inventors thus identifiedsequences of fragments of SEQ ID No 1 that match with the m/zmeasurements.

The results are shown herebelow:

m/z Rt Identified SEQ ID (z = 1) (min) Sequences N^(o) CompetenceA-Natural 1180,6434  45,83 IAILPYFAGCL 7 YES peptides LIAILPYFAGC 8 NT1067,5594 42,58 AILPYFAGCL 9 NT IAILPYFAGC 10 NT  996,5223 40,58ILPYFAGCL 11 NT B-Alkylated 1237,6649 42,98 IAILPYFAGC*L - NO peptidesLIAILPYFAGC - NT 1124,5808 39,72 AILPYFAGC*L - NT IAILPYFAGC*L - NT1053,5437 37,66 ILPYFAGC*L - NT NT, not tested *alkylated form of thecysteine

At this step, peptide ion fragmentation was not possible due to theweakness of the mass spectrometry signals. In order to confirm thepresence of a cysteine residue in the different fragments, alkylation ofthe supernatants of the deleted and the overproducing strains wasperformed with iodoacetamide with the following protocol: 150 μl of NaOH5M were added to 30 ml of supernatant. Alkylation was performed withiodoacetamide at a final concentration of 20 mM for 30 min in the dark.Samples were enriched in peptides as described above with the followingmodification, 50 μl of formic acid was added to adjust the pH to 6.5after the ultrafiltration step.

Masses corresponding to alkylated forms of all fragments (B-alkylatedpeptides) were detected, with the expected shift in HPLC retentiontimes, in the supernatant of the overproducing strain and not detectedin the deleted strain confirming that these fragments contain a cysteineamino acid.

Alkylation increased the peptide signals in mass spectrometry andallowed the fragmentation of the longer one. Fragmentation of thepeptide validated the IAILPYFAGCL sequence of the 1237,6649 mass (SEQ IDNo 7).

In conclusion, the inventors have shown that SEQ ID No 1 is theprecursor of peptides that are secreted in the supernatant of theoverproducing S. thermophilus LMD9 strain. The inventors identified 3masses corresponding to 5 different fragments of SEQ ID No 1. Theinventors validated the amino acids sequence SEQ ID No 7 (IAILPYFAGCL),which is an active competence peptide. The inventors have also showedthat the shorter sequences are products of degradation of SEQ ID No 7 byproteases and/or aminopeptidases.

12) Biologic Activity of Fragments of SEQ ID No 1

The inventors tested the activity of fragments of SEQ ID No 1 using astrain deleted for the papR gene using the protocol described in part 8.The inventors thus synthesized different fragments of SEQ ID No 1 inorder to test their biological activity. Fragments were synthesized asbelow:

SEQ ID N^(o) 7 IAILPYFAGCL SEQ ID N^(o) 12 LPYFAGCL SEQ ID N^(o) 13PYFAGCL

To test the biological activity of those fragments, peptides were addedto a culture of a strain deleted for the papR gene at a finalconcentration of 1 μM, 1 hour after the dilution of the culture at anOD₆₀₀ of 0.05 in a chemical defined medium (CDM). Transformation assayswere performed with 100 μl of cells with 1 μg of plasmid and orchromosomal DNA.

The results are disclosed herebelow.

The numbers of transformants obtained with 100 μl of competent cells areas follow:

Transforming SEQ ID SEQ ID SEQ ID DNA N^(o) 7 N^(o) 12 N^(o) 13Chromosomal 24 41 0 Plasmid 284 10² 780 10² 0

Cysteyl residue is a reactive amino acid due to the presence of a freeSH. In order to test the significance of this residue in the biologicalactivity of the peptide, SEQ ID No 7 was alkylated, as described above,and its activity was tested.

The numbers of transformants obtained with 100 μl of competent cells areas follow:

Transforming SEQ ID Alkylated DNA N^(o) 7 SEQ ID N^(o) 7 Chromosomal 350 Plasmid 70 10² 2

These results indicate that SEQ ID No 7 needs to have a free cysteylresidue to be active and that the oxydo-reduction state or thealkylation of the peptide influence the competence rate of S.thermophilus strains.

S. thermophilus strain LMG18311 is naturally poorly competent and strainCNRZ1066 is naturally not competent. However, complementation of bothstrains with a plasmid that overexpresses the papR gene makes themcompetent. The inventors tested the effect of the addition of SEQ ID No7 on the competence of both strains in the same condition as describedabove for the LMD9 strain.

The numbers of transformants obtained with 100 μl of competent cells areas follow:

Transforming LMG18311 CNRZ1066 DNA No Fragment SEQ ID N^(o) 7 NoFragment SEQ ID N^(o) 7 Chromosomal 0 477 0 0 Plasmid 7 731 10³ 0 27410³

These results indicate that the addition of SEQ ID No 7 in a culture ofS. thermophilus strain CNRZ1066 or LMG18311 renders them competent.

The inventors have thus shown that SEQ ID No 1 and fragments of SEQ IDNo 1 (particularly SEQ ID No 7) are capable of stimulating competence inStreptococcus.

1-17. (canceled)
 18. An isolated polypeptide comprising the amino acidsequence of SEQ ID No. 7 (IAILPYFAGCL), a derivative thereof having apercentage of identity of at least 70% with the amino acid sequence SEQID No. 7; or a fragment of SEQ ID No. 7, wherein said derivative orfragment is capable of stimulating competence in Streptococcus.
 19. Theisolated polypeptide of claim 18 wherein the derivative thereof has apercentage of identity of at least 95% with the amino acid sequence ofSEQ ID No.
 7. 20. The isolated polypeptide of claim 18 wherein saidpolypeptide consists of the amino acid sequence of SEQ ID No. 7 orconsists of a derivative thereof having a percentage of identity of atleast 70% with the amino acid sequence of SEQ ID No.
 7. 21. The isolatedpolypeptide of claim 22 wherein the derivative thereof has a percentageof identity of at least 95% with the amino acid sequence of SEQ ID No.7.
 22. The isolated polypeptide of claim 18 wherein said polypeptidecomprises the amino acid sequence of SEQ ID No. 1(LKTLKIFVLFSLLIAILPYFAGCL), a derivative thereof having a percentage ofidentity of at least 70% with the amino acid sequence SEQ ID No. 1; or afragment of SEQ ID No. 1, wherein said derivative or fragment is capableof stimulating competence in Streptococcus.
 23. The isolated polypeptideof claim 22 wherein the derivative thereof has a percentage of identityof at least 95% with the amino acid sequence of SEQ ID No
 1. 24. Theisolated polypeptide of claim 18, wherein the length of said isolatedpolypeptide is less than 100 amino acids.
 25. The isolated polypeptideof claim 18, further comprising an amino acid sequence corresponding toa signal peptide.
 26. An isolated nucleic acid encoding the isolatedpolypeptide as defined in claim
 18. 27. A vector comprising the nucleicacid as defined in claim 26 operably linked to a gene expressionsequence.
 28. A host cell genetically engineered with the vector asdefined in claim
 27. 29. A culture medium comprising an effective amountof the isolated polypeptide of claim 18 and nutrients for growth of abacterium of the phylum Firmicutes.
 30. The culture medium of claim 29,wherein said effective amount is between 0.1 ng/ml and 1 mg/ml.
 31. Theculture medium of claim 29 wherein the bacterium is of the genusStreptococcus.
 32. The culture medium of claim 29 wherein the bacteriumis of the species Streptococcus thermophilus.
 33. A method of producingtransformation competent bacteria of the phylum Firmicutes comprisingthe step of contacting said bacteria with an effective amount of thepolypeptide of claim
 18. 34. The method of claim 33 wherein the bacteriaare of the genus Streptococcus.
 35. A method of claim 34 wherein saidcontacting step comprises the steps of (i) culturing said bacteria in apeptide-free medium to an OD₆₀₀ between 1.5 and 2.5 and (ii) dilutingthe culture of step (i) to an OD₆₀₀ between 0.01 and 0.1, wherein saidbacteria produce said polypeptide during step (i).
 36. The method ofclaim 35 wherein the bacteria are of the species Streptococcusthermophilus.
 37. The method of claim 33 wherein said method isperformed in a culture medium comprising an effective amount of saidpolypeptide.
 38. A method for producing a mutant bacterium of the phylumFirmicutes which comprises the steps of: (a) producing transformationcompetent bacteria of the phylum Firmicutes by the method of claim 33;and (b) contacting said transformation competent bacteria withhomologous DNA under conditions to allow transformation of said bacteriawith said homologous DNA.
 39. The method of claim 38 further comprisesthe steps of selecting and/or amplifying the mutant bacteria thusgenerated.
 40. A method for identifying a compound stimulatingcompetence in a bacterium of the genus Streptococcus comprising thesteps of: i) contacting, with said compound, a host cell transformedwith a nucleic acid comprising a nucleic acid sequence coding for areporter protein under the control of all or part of a promoter precededby the inverted repeat sequence recognized by the PlcR-like regulator:(SEQ ID N^(o) 3) ATAGTGACATATATGTCTCTAT or (SEQ ID N^(o) 4)GTGGTGACATAAATGTCACTAT; and

ii) selecting the compound that stimulates the expression of saidreporter protein.
 41. The method of claim 40 wherein the host cell is abacterial cell.
 42. The method of claim 40 wherein the reporter proteinis GFP or beta-galactosidase.